The Raf-1 protein kinase functions as a critical regulator of cellular responses to multiple distinct extracellular signals. These include the proliferative response of epithelial cells to PDGF and the development of embryonic mesoderm in response to FGF. In rat PC12 cells, an adrenal phenochromocytoma cell line used as a model for neuronal development, Raf mediates both proliferation in response to EGF, and differentiation into a sympathetic neuronal phenotype in response to NGF. Raf-1 plays an important role during Drosophila and C. elegans development and in the frog, Xenopus laevis, Raf-1 mediates posterior mesoderm formation. Oncogenic Raf-1 has been implicated as an etiologic factor in several human cancers including osteosarcoma and small cell lung carcinomas. Raf-1 function is required to mediate cellular transformation by a number of other oncogenes. Raf-1 is a key mediator of numerous pathologic and physiological processes. Despite the recent progress in understanding the activation of Raf-1 in growth factor-stimulated signal transduction, relatively little is understood about the molecular mechanisms that negatively regulate Raf-1 protein kinase activity in response to extracellular signals. There is evidence that Raf activity may be negatively regulated by a wide range of physiological stimuli which control cAMP and calcium levels. The cAMP-dependent protein kinase (PKA) has been shown to regulate Raf-1 activation. The recent studies have demonstrated that Raf-1 activity is regulated by PKA at two levels: i) prevention of Raf-1 activation, and ii) inhibition of Raf-1 catalytic activity. Similarly, they have found that a mediator of calcium signaling, calcium/calmodulin dependent kinase II (CaMK) can also regulate Raf-1 through inhibition of catalytic activity. Thus, agents which increase intracellular calcium levels, like those which increase cAMP, appear to be important regulators of growth factor stimulated proliferation and differentiation mediated by Raf-dependent signaling pathways. In the studies described in this application, they will elucidate the molecular mechanisms involved in the post-translational negative regulation of Raf proto-oncogene activity following activation of cAMP and calcium dependent processes. These studies may help to shed light on the molecular mechanisms involved in coordinately regulating cellular responses to receptor-mediated signaling events. In addition to adding to our understanding of the regulatory pathways which impinge on Raf in vivo, these studies may provide novel information for the design of therapeutic intervention in the treatment of certain human cancers.